Elsevier

Aquatic Toxicology

Volume 126, 15 January 2013, Pages 326-337
Aquatic Toxicology

Molecular mechanisms associated with increased tolerance to the neonicotinoid insecticide imidacloprid in the dengue vector Aedes aegypti

https://doi.org/10.1016/j.aquatox.2012.09.010Get rights and content

Abstract

Mosquitoes are vectors of several major human diseases and their control is mainly based on the use of chemical insecticides. Resistance of mosquitoes to organochlorines, organophosphates, carbamates and pyrethroids led to a regain of interest for the use of neonicotinoid insecticides in vector control. The present study investigated the molecular basis of neonicotinoid resistance in the mosquito Aedes aegypti. A strain susceptible to insecticides was selected at the larval stage with imidacloprid. After eight generations of selection, larvae of the selected strain (Imida-R) showed a 5.4-fold increased tolerance to imidacloprid while adult tolerance level remained low. Imida-R larvae showed significant cross-tolerance to other neonicotinoids but not to pyrethroids, organophosphates and carbamates. Transcriptome profiling identified 344 and 108 genes differentially transcribed in larvae and adults of the Imida-R strain compared to the parental strain. Most of these genes encode detoxification enzymes, cuticle proteins, hexamerins as well as other proteins involved in cell metabolism. Among detoxification enzymes, cytochrome P450 monooxygenases (CYPs) and glucosyl/glucuronosyl transferases (UDPGTs) were over-represented. Bioassays with enzyme inhibitors and biochemical assays confirmed the contribution of P450s with an increased capacity of the Imida-R microsomes to metabolize imidacloprid in presence of NADPH. Comparison of substrate recognition sites and imidacloprid docking models of six CYP6s over-transcribed in the Imida-R strain together with Bemisia tabaci CYP6CM1vQ and Drosophila melanogaster CYP6G1, both able to metabolize imidacloprid, suggested that CYP6BB2 and CYP6N12 are good candidates for imidacloprid metabolism in Ae. aegypti. The present study revealed that imidacloprid tolerance in mosquitoes can arise after few generations of selection at the larval stage but does not lead to a significant tolerance of adults. As in other insects, P450-mediated insecticide metabolism appears to play a major role in imidacloprid tolerance in mosquitoes.

Highlights

► A strain of Aedes aegypti was selected at the larval stage with imidacloprid. ► The selected strain showed a 5.4-fold increased tolerance to imidacloprid at G8. ► 344 genes were differentially transcribed in larvae from the resistant strain. ► 24 P450s showed a significant differential transcription in larvae or adults. ► Imidacloprid docking models suggest that 2 P450 may contribute to resistance.

Introduction

Mosquitoes transmit numerous human and animal diseases and their control represents a public health challenge worldwide. Dengue fever and yellow fever viruses are both transmitted by the mosquito Aedes aegypti. Fifty million people have been estimated to be affected by dengue fever with nearly 2.5 billion people at risk while 30,000 deaths are attributed to yellow fever each year (Tomori, 2004). Because vaccination against dengue is not available and access to yellow fever vaccine is not effective worldwide (Griffiths et al., 2010, Trent et al., 2010), limiting the transmission of these diseases is highly dependent on controlling vector populations (Hemingway et al., 2006).

Effective vector control generally relies on the use of chemical insecticides targeting adults or larvae (Killeen et al., 2002). However, resistance of mosquitoes to all classes of chemical insecticides has been reported and threatens vector control programs (Hemingway and Ranson, 2000). Resistance to insecticides can be the consequence of a mutation of the protein targeted by the insecticide (target-site resistance), a lower penetration or a sequestration of the insecticide, or an increased biodegradation of the insecticide (metabolic resistance) (Hemingway et al., 2004). Detoxification enzymes such as cytochrome P450 monooxygenases (P450s or CYPs), glutathione S-transferases (GSTs) and carboxy/choline esterases (CCEs) are well known for their role in the metabolism of insecticides in insects (Scott, 1999, Li et al., 2007) and over-production of these enzymes has been associated with resistance to all classes of chemical insecticides in mosquitoes (Hemingway et al., 2004).

The increasing resistance level of mosquitoes to organochlorines (OCs), organophosphates (OPs), carbamates (Carbs) and pyrethroids (Pyrs) led to a renewed interest for the use of neonicotinoids against mosquitoes (Paul et al., 2006, Pridgeon et al., 2008). Imidacloprid ((E)-1-(6-chloro-3-pyridinylmethyl)-N-nitroimidazolidin-2-ylideneamine) is a neonicotinoid insecticide targeting acetylcholine receptors in insect nervous systems (Tomizawa and Casida, 2005). This insecticide is extensively used in agriculture against pests of various crops such as cotton, cereals and vegetables (Mullins, 1993, Elbert et al., 2008). Several studies conducted on agricultural pests suggested the capacity of several insect species to develop resistance to imidacloprid and revealed that resistance to neonicotinoids was linked to higher levels of P450s (Karunker et al., 2008, Feng et al., 2010, Puinean et al., 2010a). In addition, other studies have demonstrated the capacity of Drosophila melanogaster CYP6G1 (DmCYP6G1) and Bemisia tabaci CYP6CM1vQ (BtCYP6CM1vQ) to metabolize imidacloprid (Jouben et al., 2008, Karunker et al., 2009). However, despite the potential use of neonicotinoids for vector control, resistance mechanisms of mosquitoes to these insecticides remain poorly investigated.

In this study, a laboratory strain of Ae. aegypti susceptible to insecticides was selected with imidacloprid at the larval stage. Tolerance to imidacloprid increased five times in larvae after eight generations of selection. The potential mechanisms responsible for this increased tolerance were investigated using a combination of transcriptomic and biochemical approaches. Several candidate genes belonging to detoxification enzymes and other protein families were identified as potentially involved in imidacloprid tolerance. As P450s appear to play a major role, comparison of protein sequences and insecticide docking predictions were used to identify several candidate Ae. aegypti P450s for imidacloprid metabolism. These results are discussed in regards of known and new potential insecticide resistance mechanisms in insects.

Section snippets

Selection procedure

Mosquitoes were reared in standard insectary conditions (26 °C, 14 h/10 h light/dark period, 80% relative humidity) in tap water (larvae) and net cages (adults). Larvae and adults were fed with hay pellets and papers impregnated with honey, respectively. Blood feeding of adult females was performed on mice. The laboratory strain Bora-Bora, originating from French Polynesia, was used as a parental strain for selection experiments. This strain is susceptible to all insecticides and does not present

Comparative bioassays

Larval bioassays (Table 1) performed after eight generations of selection on G9 individuals revealed an increased tolerance to imidacloprid of the Imida-R strain compared to the parental susceptible strain (TR50 of 5.4-fold). Imida-R larvae showed cross-tolerance to other neonicotinoids (thiamethoxam, TR50 of 4.4 and acetamiprid, TR50 of 3.55) and in a lesser extent to DDT (TR50 of 1.8) while no cross-tolerance to permethrin, propoxur and temephos was detected (Table 2).

Adult topical bioassays

Tolerance level in the Imida-R strain

Since neonicotinoid insecticides have a mode of action different from other chemical insecticides mostly used for vector control (pyrethroids, OCs, OPs and carbamates), they have been suggested as a possible alternative to manage insecticide resistance in the field (Paul et al., 2006, Tiawsirisup et al., 2007, Pridgeon et al., 2008). In this context, the present study aimed at investigating molecular mechanisms associated with increased tolerance to the neonicotinoid insecticide imidacloprid in

Conclusions

The present study provides new insights about molecular mechanisms associated with neonicotinoid tolerance in mosquitoes and other insects. Our results reveal that imidacloprid increased tolerance in mosquitoes can arise after few generations of selection at the larval stage but did not lead to a significant tolerance of adults, suggesting that the selected tolerance mechanisms are life-stage specific. Larval increased tolerance to imidacloprid was associated to important modifications of gene

Data deposition

The description of the microarray used in this study can be accessed at ArrayExpress (http://www.ebi.ac.uk/arrayexpress) accession no. A-MEXP-1966.

All experimental microarray data can be accessed at VectorBase (http://VectorBase.org) and ArrayExpress database accession no. E-MTAB-616

Acknowledgments

The present research project was funded by the French National Research Agency (ANR, project 07SEST014 MOSQUITO-ENV). M.A. Riaz was funded by the Higher Education Commission (HEC) of Pakistan and the French ‘Ministère de l’Education Supérieure et de la Recherche’. We thank Jessica Roux, Sylvie Veyrenc and Thierry Gaude for technical assistance. We thank Dr. S. Morin and Dr. R. Sertchook for providing CYP6CM1vQ P450 model. We thank Dr. R. MacCallum and Vectorbase for technical support with

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